This invention relates generally to immunoassays. More particularly the invention relates to an assay which allows for detection of a specific strain of a disease related conformational form of a protein (such as PrPSc) which may have very low antibody binding affinity.
Prions are infectious pathogens that cause invariably fatal prion diseases (spongiform encephalopathies) of the central nervous system in humans and animals. Prions differ significantly from bacteria, viruses and viroids. The dominating hypothesis is that no nucleic acid is necessary to allow for the infectivity of a prion protein to proceed.
A major step in the study of prions and the diseases they cause was the discovery and purification of a protein designated prion protein [Bolton, McKinley et al. (1982) Science 218:1309-1311; Prusiner, Bolton et al. (1982) Biochemistry 21:6942-6950; McKinley, Bolton et al. (1983) Cell 35:57-62]. Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. PrPC is encoded by a single-copy host gene [Basler, Oesch et al. (1986) Cell 46:417-428] and when PrPC is expressed it is generally found on the outer surface of neurons. Many lines of evidence indicate that prion diseases results from the transformation of the normal form of prion protein (PrPC) into the abnormal form (PrPSc). There is no detectable difference in the amino acid sequence of the two forms. However, PrPSc when compared with PrPC has a conformation with higher xcex2-sheet and lower xcex1-helix content [Pan, Baldwin et al. (1993) Proc Natl Acad Sci USA 90:10962-10966; Safar, Roller et al. (1993) J Biol Chem 268:20276-20284]. The presence of the abnormal PrPSc form in the brains of infected humans or animals is the only disease-specific diagnostic marker of prion diseases.
PrPSc plays a key role in both transmission and pathogenesis of prion diseases (spongiform encephalopathies) and it is a critical factor in neuronal degeneration [Prusiner (1997) The Molecular and Genetic Basis of Neurological Disease, 2nd Edition: 103-143]. The most common prion diseases in animals are scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle [Wilesmith and Wells (1991) Curr Top Microbiol Immunol 172:21-38]. Four prion diseases of humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Streussler-Sheinker Disease (GSS), and (4) fatal familial insomnia (FFI) [Gajdusek (1977) Science 197:943-960; Medori, Tritschler et al. (1992) N Engl J Med 326:444-449]. Initially, the presentation of the inherited human prion diseases posed a conundrum which has since been explained by the cellular genetic origin of PrP.
Prions exist in multiple isolates (strains) with distinct biological characteristics when these different strains infect in genetically identical hosts [Prusiner (1997) The Molecular and Genetic Basis of Neurological Disease, 2nd Edition: 165-186]. The strains differ by incubation time, by topology of accumulation of PrPSc protein, and in some cases also by distribution and characteristics of brain pathology [DeArmond and Prusiner (1997) Greenfield""s Neuropathology, 6th Edition:235-280]. Because PrPSc is the major, and very probably the only component of prions, the existence of prion strains has posed a conundrum as to how biological information can be enciphered in a molecule other than one comprised of nucleic acids. The partial proteolytic treatment of brain homogenates containing some prion isolates has been found to generate peptides with slightly different electrophoretic mobilities [Bessen and Marsh (1992) J Virol 66:2096-2101; Bessen and Marsh (1992) J Gen Virol 73:329-334; Telling, Parchi et al. (1996) Science 274:2079-2082]. These findings suggested different proteolytic cleavage sites due to the different conformation of PrPSc molecules in different strains of prions. Alternatively, the observed differences could be explained by formation of different complexes with other molecules, forming distinct cleavage sites in PrPSc in different strains [Marsh and Bessen (1994) Phil Trans R Soc Lond B 343:413-414]. Some researchers have proposed that different prion isolates may differ in the glycosylation patterns of prion protein [Collinge, Sidle et al. (1996) Nature 383:685-690; Hill, Zeidler et al. (1997) Lancet 349:99-100]. However, the reliability of both glycosylation and peptide mapping patterns in diagnostics of multiple prion strains is currently still debated [Collings, Hill et al. (1997) Nature 386:564; Somerville, Chong et al. (1997) Nature 386:564].
A system for detecting PrPSc by enhancing immunoreactivity after denaturation is provided in Serban, et al., Neurology, Vol. 40, No. 1, Ja 1990. Sufficiently sensitive and specific direct assay for infectious PrPSc in biological samples could potentially abolish the need for animal inoculations completely. Unfortunately, such does not appear to be possible with current PrPSc assaysxe2x80x94it is estimated that the current sensitivity limit of proteinase-K and Western blot-based PrPSc detection is in a range of 1 xcexcg/ml which corresponds to 104-105 prion infectious units. Additionally, the specificity of the traditional proteinase-K-based assays for PrPSc is in question in light of recent findings of only relative or no proteinase-K resistance of undoubtedly infectious prion preparations [Hsiao, Groth et al. (1994) Proc Natl Acad Sci USA 91:9126-9130] Telling, et al. (1996) Genes and Dev. 
Human transthyretin (TTR) is a normal plasma protein composed of four identical, predominantly xcex2-sheet structured units, and serves as a transporter of hormone thyroxine. Abnormal self assembly of TTR into amyloid fibrils causes two forms of human diseases, namely senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP) [Kelly (1996) Curr Opin Strut Biol 1):11-7]. The cause of amyloid formation in FAP are point mutations in the TTR gene; the cause of SSA is unknown. The clinical diagnosis is established histologically by detecting deposits of amyloid in situ in biopsy material.
To date, little is known about the mechanism of TTR conversion into amyloid in vivo. However, several laboratories have demonstrated that amyloid conversion may be simulated in vitro by partial denaturation of normal human TTR [McCutchen, Colon et al. (1993) Biochemistry 32(45):12119-27; McCutchen and Kelly (1993) Biochem Biophys Res Commun 197(2) 415-21]. The mechanism of conformational transition involves monomeric conformational intermediate which polymerizes into linear xcex2-sheet structured amyloid fibrils [Lai, Colon et al. (1996) Biochemistry 35(20):6470-82]. The process can be mitigated by binding with stabilizing molecules such as thyroxine or triiodophenol [Miroy, Lai et al. (1996) Proc Natl Acad Sci USA 93(26): 15051-6].
In view of the above points, there is clearly a need for a specific, high flow-through, and cost-effective assay for testing sample materials for the presence of specific strains of a pathogenic protein including transthyretin and prion protein.
Assay methodology of the invention allows for: (1) determining if a sample contains a conformation of a protein which is associated with disease and the concentration and amount of such if present; (2) determining the amount of a chemical compound such as a protease resistant disease related protein in a sample and by subtracting that amount from the total amount of disease related protein present determining the amount of protease sensitive disease protein in the sample; and (3) determining the strain and incubation time of a disease related protein by (i) relating the relative amounts of protease resistant and protease sensitive protein to known strains to thereby determine the strain; and (ii) plotting the concentration of protease sensitive protein on a graph of incubation time versus concentration of protease sensitive protein for known strains to predict the incubation time of a given unknown strain.
The presence and concentration of protein in a disease related conformation is determined via one of three different basic methods. Pursuant to the primary method a sample first is divided into two portions. A first portion is contacted with a labeled antibody which binds to the non-disease conformation of the protein but not to the disease related conformation of the protein. The second portion is then subjected to a protein unfolding treatment which increases the binding affinity for any protein in the second conformation for the antibody. In general the protein unfolding treatment will expose an epitope to which the antibody can bind which epitope is unexposed in the disease related conformation. Accordingly, disease related protein which did not bind the antibody prior to the protein unfolding treatment will now bind the antibody. A comparison is then made between the amount of antibody binding to protein in the first untreated portion with the amount of antibody binding to protein in the second portion. A difference between the amount of antibody binding in the first and second portions indicates the presence of disease related protein in the sample. Depending on the protein and the protein unfolding treatment used it may be necessary to make an adjustment due to the effect of the treatment one protein in the non-disease related conformation.
Stated in a step-by-step manner the basic assay method of the invention comprises (a) providing a sample suspected of containing a protein (having a first conformation and a second, disease-related conformation), (b) dividing the sample into first and second portions, (c) contacting the first portion with an antibody that binds to the first conformation with higher affinity than to the second conformation, (d) subjecting the second portion to a protein unfolding treatment to cause any protein in the second conformation to adopt a different conformation having a higher affinity for the antibody, (e) contacting the second portion with the antibody, (f) determining the relative levels of antibody binding to said first and second portions, and (g) determining the presence or absence of protein in the second conformation based on the comparison.
Once it has been determined that a sample contains protein in disease related conformation it is further useful to determine the strain in relationship to incubation time of the protein. This can be done by obtaining another portion of the sample which tested positive for protein in the disease related conformation. This portion is subjected to limited protease treatment which hydrolyzes most protein in the sample but for highly resistant protein in the disease related conformation, e.g. protease resistant PrP 27-30. The concentration and amount of the treatment resistant protein is then determined. By subtracting the amount of treatment resistant protein in the sample from the total amount of disease related protein in the sample one obtains the amount of disease related protein in the sample which is sensitive to protease digestion, e.g. protease sensitive PrPSc. Each strain of disease related protein has a known ratio of total protein in the disease related conformation (e.g. native PrPSc) to amount of protein in disease related conformation which is denatured by a protein denaturing treatment (protease sensitive PrPSc). Thus, the (total: denatured) ratio can be matched to that of a known strain to determine the strain in a sample.
The method of determining the strain of any disease conformation of a protein can also be stated in a step-by-step fashion. The method is carried out by (a) isolating protein in the disease related formation, e.g. by centrifugation; (b) treating the isolated protein with a compound (e.g. guanidine hydrochloride) which denatures one form of the isolated protein but not another; (c) determining the ratio of protein resistant to the treatment relative to the total amount of disease related protein; and (d) comparing the ratio to that of a predetermined standard of a known strain thereby determining the strain of disease related protein in a sample This method is more readily applied when the strain found matches to a known strain. If there is no match to a known strain and no experimental error was made, it may be assumed that a new strain has been discovered.
The incubation time of a disease related conformation of a protein can be determined even if the strain is previously unknown. This is determined by (a) isolating protein in the disease related formation, e.g. by centrifugation; (b) treating the isolated protein (e.g. with proteinase K) which hydrolyzes one form of the isolated protein but not another; (c) subtracting the amount of disease related protein not denatured (treatment resistant protein) from the total amount of disease related protein to find the amount of disease related protein which is denatured; (d) plotting the amount of disease related (treatment sensitive) protein found in (c) on a graph of incubation time versus amount of disease related protein not denatured (which graph has plotted known strains with known incubation times); and (e) thereby predict the incubation time.
The assay of the invention is useful in assaying samples which contain proteins which are present in at least two conformations (e.g., a native non-disease conformation and a disease conformation) and are present at levels of 1xc3x97103 particles/ml or less. The present invention utilizes antibodies which do not bind or have a relatively low degree of affinity for the tightly configured disease-conformation of the protein. One useful antibody for binding to PrPC is the monoclonal antibody 3F4 produced by the hybridoma cell line ATCC HB9222 deposited on Oct. 8, 1986 in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 and disclosed and described in U.S. Pat. No. 4,806,627 issued Feb. 21, 1989xe2x80x94incorporated by reference to disclose antibodies which selectively bind PrPC. In addition to antibody other binding partners which bind the non-disease related conformation but not the disease related conformation could be used in the assay of the invention. Antibodies such as 3F4 and others used in the assays described in the examples are commercially available.
To demonstrate the primary method behind the present invention one begins with a starting sample which is divided into at least two portions. The first portion is contacted with labeled antibodies without treating the proteins and the second portion is treated with labeled antibodies after the proteins have been subjected to a protein unfolding treatment which causes any proteins in the disease conformation to assume a conformation which has a higher degree of binding affinity for the antibodies (e.g. exposes a previously unexposed epitope). The readings are compared (i.e., one subtracted from the other) and the presence of proteins in the disease related conformation are deduced based on the difference between the two readings.
Pursuant to a second embodiment of the basic assay, it is possible to utilize the basic concept behind the present invention without obtaining two readings for each assay. This can be done by establishing a standard based on carrying out the assay on a statistically significant number of closely related samples. After the standard has been established one will know the level of antibody binding which should be observed when a given sample does not contain any proteins in the disease related conformation. Using the standard, one then subjects a sample to be tested to a protein unfolding treatment so as to convert any proteins in the disease related conformation to a different conformation which has a much higher degree of binding affinity for the label antibodies. The measurement obtained is then compared with the standard. If the difference between the standard and the measurement obtained is outside of a given range it can be deduced that the original sample included proteins in the disease related conformation. These results could be confirmed (1) via the first embodiment described above and/or (2) by testing the sample with an antibody which binds only the disease related conformation of the proteinxe2x80x94see US96/14840.
A third embodiment of the invention can utilize either of the embodiments disclosed above along with the formulae provided herein in order to calculate (quantitatively) the number of proteins (concentration) in the disease related conformation present within the original sample.
In accordance with any of the assay embodiments it is preferable to pre-treat the sample being tested to (1) remove as many contaminant proteins as possible, and (2) increase the concentration of disease related protein in the sample relative to the non-disease related conformation of the protein. For example, the initial sample can be chemically treated with a compound which preferentially degrades or denatures contaminant proteins and/or the relaxed, non-disease form of the protein and/or is exposed to antibodies which preferentially bind to (in order to remove) contaminants and/or non-disease conformation of the protein.
It may be possible to enhance further the sensitivity of various aspects of the invention by concentrating the disease conformation of a protein by adding a compound which selectively binds to the disease conformation to form a complex and centrifuging the sample to precipitate out the complex which is then tested in accordance with the methods described here. Specifics regarding such concentration methods are described in detail in our co-pending application Ser. No. 09/026,967 entitled xe2x80x9cProcess for Concentrating Protein with Disease-Related Conformationxe2x80x9d.
The different embodiments of the assay of the invention described above are all xe2x80x9cdirectxe2x80x9d types of immunoassaysxe2x80x94meaning that the sample is directly assayed with the labeled antibody either with or without treatment to change the conformation of any disease related conformation proteins present in the sample. An xe2x80x9cindirectxe2x80x9d assay may also be used. For example, it may be desirable to enhance the number of disease related proteins in the sample (if any) by the use of a transgenic mouse and thereby enhance any signal obtained. To carry out these embodiments of the invention, the sample is first used to inoculate a transgenic mouse which has had its genome modified so that it will develop symptoms of disease when inoculated with proteins in the disease related conformation. After the mice are inoculated, a sufficient period of time is allowed to pass (e.g., 30 days) after which the transgenic animal is sacrificed and a sample such as homogenized brain tissue from the mouse is used in the direct assay described above. The present invention enhances the ability of transgenic mice to detect prions by shortening the period of time which must pass until a determination can be made as to whether the original sample included proteins in the disease related conformation. It would also be possible to use mice of the type disclosed and described in any of U.S. Pat. Nos. 5,565,186; 5,763,740; or 5,792,901 or to apply epitope tagged PrP as disclosed in U.S. Pat. No. 5,750,361 (incorporated by reference) to affinity purify the PrPSc from the brain of a Tg mouse and thereafter apply the assay of the present invention. Without the present invention the mouse is inoculated and one must wait until the inoculated mouse actually demonstrates symptoms of the disease. Depending on the mouse, this can take several months or even years. Any of the assays of the present invention could be used with any transgenic mice such as those described above. The assay could be used well before the mouse developed symptoms of disease thereby shortening the time needed to determine if a sample includes infectious proteins.
The assay methodology of the present invention can be applied to any type of sample when the sample is suspected of containing a protein which occurs in at least two conformations. The protein must occur in one conformation which binds to known antibodies, antibodies which can be generated or other specific binding partners. The second conformation must be sufficiently different from the first conformation in terms of its binding affinity so that the two conformations can be distinguished by using antibodies or binding partners which have a much higher degree of affinity for the first conformation than for the second conformation. In its conceptually simplest form, the invention works best when a known labeled antibody binds to a non-disease form of a protein with a high degree of affinity, and does not bind (or binds with an extremely low degree of affinity) to the same protein when it is present in its disease related conformation. However, in reality, a given protein may have more than two conformations. The protein may have more than one non-disease conformation and more than one disease related conformation, (Telling, et al., Science (1996)). The invention is still useful when multiple conformations of non-disease and disease forms of the protein existxe2x80x94provided that (1) at least one non-disease conformation differs from at least one disease conformation in terms of its binding affinity; and (2) it is possible to treat the disease related conformation of the protein so as to substantially enhance its binding affinity.
As indicated above, the assay of the invention can be used to assay any type of sample for any type of protein, provided the protein includes a non-disease and a disease related conformation. However, the invention was particularly developed to assay samples for the presence of (1) PrP proteins and determine whether the sample included a PrP protein in its disease conformation, i.e., included PrPSc (2) insoluble forms of xcex2A4 associated with Alzheimer""s disease and (3) transthyretin. Accordingly, much of the following disclosure is directed to using the immunoassay of the present invention to detect the presence of either PrPSc (or to a lesser degree xcex2A4 or transthyretin (TTR)) in a samplexe2x80x94it being understood that the same general concepts are applicable to detecting disease related conformations of a wide range of different types of proteins. Further, the disclosure is particularly directed to describing how to determine the incubation time and the particular strain of infectious prions (PrPSc) in a samplexe2x80x94it being understood that the same general concepts are applicable to determining the incubation time and particular strain of other constricted proteins associated with different diseases.
The present method of PrPSc detection was developed by labeling selected purified IgG with Europium. Antibodies used (3F4) have a high binding affinity for PrPC (non-disease conformation) which comprises an xcex1-helical rich conformation. The antibodies have a low binding affinity for PrPSc (disease conformation) which comprises a xcex2-sheet rich conformation. The IgG may be obtained from common monoclonal, polyclonal, or recombinant antibodies, typically recognizing the sequence 90-145 of PrPC and conformationally unfolded prion protein. Different conformations of recombinant prion protein were chemically crosslinked to polystyrene plates through a glutaraldehyde activation step. The relative affinities of the Eu-labeled IgG with xcex1-helical, xcex2-sheet, and random coil conformation of recombinant Syrian hamster prion protein corresponding to sequence 90-231 were determined by time-resolved, dissociation-enhanced fluorescence in a 96-well polystyrene plate format.
A determination of the relative affinities of different labeled antibodies for proteins can be made by different methods. However, the disease related conformation of a protein is often present in a very low concentration relative to that of the non-disease conformation. Accordingly, it often requires very sensitive methods to detect any increase caused by the treatment of the disease conformation of the protein. Time-resolved, dissociation-enhanced fluorescence and more preferably dual wavelength, laser-driven fluorometers are particularly useful devicesxe2x80x94see Hemmilxc3xa4 et al., Boianalytical Applications of Labeling Technologies (eds. Hemmilxc3xa4) 113-119 (Wallas Oy. Turku, Finland, 1995).
By carefully calibrating this method, it is possible to detect the signal increase in antibody reactivity in the transition from xcex2-sheet conformational state to denatured state. This signal is relatively large compared to that obtained from the transformation from its native xcex1-helical to the treated relaxed, or denatured state. Thus, the original conformational state of prion protein can be assigned by the differential assay in native and treated states. When a sample containing no xcex2-sheet rich protein is treated some increase is obtained in immunoreactivity. This amount of increase must be adjusted for and after doing such the resulting concentration or amount is referred to the xe2x80x9cadjusted amount.xe2x80x9d The amount of antibody-specific binding over that obtained for xcex1-helical conformation of PrPC (beyond the adjusted amount) is a measure of the presence of the xcex2-sheet rich conformation which is essential for pathogenicity and infectivity of PrPSc.
An aspect of the invention is to provide an immunoassay which is applicable to assaying samples containing proteins, which samples are suspected of containing a protein which occurs within a native non-disease conformation and a disease related conformation (e.g., PrP protein, xcex2A4 protein and transthyretin).
Another aspect of the invention is to provide an assay which differentiates between (1) disease related proteins or portions thereof which are not hydrolyzed by limited protease treatment with a protease such as proteinase K (protease resistant proteins, e g PrP 27-30) and (2) disease related proteins which are hydrolyzed by a limited protease treatment with a protease such as proteinase K (e g., protease-sensitive PrPSc).
Another aspect is to provide a method for determining the ratio of total native disease related protein to disease related protein which is denatured by protein denaturing treatment.
An advantage of the invention is that it allows for detection of disease related protein (e.g., protease sensitive PrPSc) which is hydrolyzed during protease digestion of the non-disease, native protein (e.g., PrPC) and thus which would not be detected via conventional Western Blot methodologies.
A feature of the invention is that it can be used to calculate the incubation time of an infectious protein.
An advantage of the present invention is that the immunoassay can quickly and accurately determine the presence of proteins in the disease related conformation (e.g., PrPSc, xcex2A4 and transthyretin) even though the antibody used in the assay does not bind or has a very low degree of binding affinity for the protein in the disease related conformation and the disease related conformation is present in a lower concentration than the non-disease conformation.
Another object of the invention is to provide an assay which makes it possible to not only determine (1) whether a pathogenic particle is present in a sample but (2) determine the concentration of the particles in a sample, (3) determine the particular strain of particle present, and (4) the incubation time.
A feature of the invention is that the signal obtained can be enhanced by the use of transgenic animals, e.g., mice which are used to detect the presence of a protein in a sample.
Another feature is that time-resolved, dissociation-enhanced fluorescence or a dual wavelength, laser driven fluorometer can be used to enhance sensitivity.
Another advantage is that the assay can detect levels of the disease causing conformation of a protein at a concentration of 1xc3x97103 particles/ml or less.
A specific object is to provide a diagnostic assay for determining the presence of infectious prion protein in variable sample materials obtained or derived from human, primate, monkey, pig, bovine, sheep, deer, elk, cat, dog, mouse, and chicken tissues and/or body fluids.
Another specific object is to provide a diagnostic assay for determining the presence of xcex2A4 protein in variable sample materials obtained or derived from human, primate, monkey, pig, bovine, sheep, deer, elk, cat, dog, mouse, and chicken tissues and/or body fluids.
Another object is to provide a rapid assay for native infectious prion protein in the brains of transgenic and non-transgenic animals injected with sample material potentially containing prions.
Another object is to provide a method to evaluate decontamination procedures by assaying the level of denaturation of pathogenic proteins (e.g., prions or xcex2-sheet xcex2A4) after such treatments.
Another object is to provide a rapid method for screening different compounds to evaluate their potential for treating diseases associated with disease conformations of different proteins such as by screening compounds for their stabilizing effect on different protein conformations (e.g., PrPC or xcex1-helical conformation of xcex2A4) or their destabilizing impact on the pathogenic conformation (e.g., PrPSc or xcex2-sheet conformation of xcex2A4) of a protein.
Another object is to provide a rapid method for screening different pharmaceutical compounds with potential for prion disease treatment such as by screening compounds for their stabilizing effect on xcex1-helical conformation of a normal isoform of the PrPC protein or their destabilizing impact on the xcex2-sheet conformation of the pathogenic isoform of the PrPSc protein.
Another advantage is that the process can be carried out without an antibody directly able to recognize an infectious conformation of a protein, and without using a proteinase K step to eliminate the signal of normal (non-disease) isoforms of the protein such as PrPC.
Another advantage is that in the invented process there is no need for the antibody directly able to recognize pathogenic conformation of xcex2A4 or transthyretin.
An important feature of the assay is the rapid, cost effective and high flow-through design which can be designed with the capacity to screen 96 samples per day per 96 well plate.
Another aspect of the invention is the diagnostic method to quantitatively detect TTR in the abnormal, amyloid conformation in sample material obtained from human and animal tissues, body fluids, and pharmaceuticals. The invented process provides a direct, sensitive method to distinguish and quantify the normal and amyloid conformations of TTR in a mixture present in sample materials.
The quantitation is based on a measurement of the difference in affinities of monoclonal or polyclonal antibodies with TTR in normal or amyloid conformation against random coil conformation. The present invention describes three methods of evaluation and the mathematical formula used for such quantification.
An important object is to provide specific diagnostic assay for pathogenic TTR in variable sample materials obtained or derived from human, primate, monkey, pig bovine, sheep, deer, elk, cat, dog, and chicken tissues.
Another object is to provide a rapid assay for amyloid form of TTR in transgenic animals.
Another object is to provide a rapid method to screen different pharmaceutical compounds with potential for treatment of senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). Such compounds are screened for their stabilizing effect on normal conformation of TTR or their destabilizing impact on the amyloid conformation of TTR
Still another object is to provide a rapid method to screen the impact of different spontaneous and designed mutations in the TTR gene on conformation, stability and amyloid formation of such TTR gene products in transgenic animals harboring natural or artificial APP genes.
The specific advantage is that invented assay may detect a pathogenic forms of TTR in a mixture with denatured nonpathogenic forms of the same or in a mixture with a soluble form of TTRxe2x80x94for example, detect less than 1xc3x97103 particles per ml.
These and other objects, advantages, and features of the invented process will become apparent to those skilled in the art upon reading the details of the assay method, antibody development and testing, and transgenic mouse as more fully described below with reference to the attached figures.